Enterococci are important causes of endocarditis, urinary tract infections and nosocomial infections. In a sense, the many antibiotic resistances of enterococci function somewhat like a virulence factor because they enable enterococci to survive and colonize while other organisms are eliminated. The recent appearance of plasmid-mediated beta-lactamase (Bla) in enterococci, described by the P.I., is medically important because this enzyme mediates resistance to the antibiotics most often used for enterococcal infections. There is also concern that this enzyme may now more easily spread into streptococci which share many resistance genes with enterococci. The DNA sequence of the enterococcal Bla (Ent-Bla) gene, determined in the P.l.'s laboratory, is identical to one and highly related to other structural genes for staphylococcal Bla (Staph-Bla); unlike most Staph-Bla's, however, Ent-Bla is constitutively produced and cell-bound, differences that are not explained by the DNA sequence. The major focus of this proposal is to investigate the differences in expression of Ent-Bla and Staph-Bla. Both Staph-Bla and Ent-Bla have now been cloned into a shuttle vector and then introduced into Staph and Ent recipients. Both enzymes are cell bound in an enterococcal host and extracellularly released in a staphylococcus. The Staph-Bla clone (taken from a wild type plasmid which encodes inducible Bla) produces very low, uninducible levels of Bla in Ent and low, marginally inducible amounts of Bla in Staph; Ent-Bla is produced constitutively and at high levels in both hosts. These and other data suggest that the Staph-Bla clone encodes repressor but lacks anti- repressor activity that was present on the wild type plasmid. MICs of the clones show a much higher MIC of penicillin for Ent-Bla than Staph-Bla in Ent, but equal MICs in Staph; this supports the possibility that a constitutive mutant is helpful for full expression of Bla in enterococci. The expression differences will be investigated initially by subcloning and transposon mutagenesis in order to identify the Staph-Bla regulatory regions, and by complementation to determine if Ent-Bla is repressible and if the Staph-Bla clone can be derepressed. Subsequently, it is planned to characterize the staphylococcal Bla repressor and the putative Bla anti-repressor (which we further hypothesize is either not present or not functional in enterococci). The genes for these regulators will be sequenced and these sequences will be compared to other sequenced genes and analyzed for potential DNA binding domains, penicillin binding sites, and transmembrane regions. Eventually, these constructs will be introduced into Ent to determine if these components can generate staphylococcal-type regulation in enterococci. This work should generate important information regarding the regulation of the staphylococcal enzyme, and should form the basis for other studies that will define the mechanism of the interactions of the various components involved in Bla induction.